SYNERGISTIC STIMULATION AND PRIMING OF NEUTROPHILS

这项提议的长期目标是阐明确切的 启动和启动过程中涉及的一系列生化事件 中性粒细胞刺激超氧化物(02-)的产生。超氧化物是 它们依赖氧气的抗菌机制的一个关键成分 对感染生物提供主要防御的细胞。一定的 细胞因子刺激中性粒细胞释放更多的02-。 这些因素的治疗价值现在正在临床上进行测试 多种癌症和艾滋病患者的治疗效果令人振奋。因此， 这里概述的实验与理解宿主有关- 防御机制和免疫缺陷疾病。 具体地说，该项目涉及两个基本上未知的领域。这些 主要内容有：(1)二十碳四烯酸的作用机理(S) 酸(HETE)在调节02-产生中，和(2)之间的联系 蛋白质磷酸化和02-生成。中性粒细胞产生5-和 15-HETE在各种情况下。5-HETE戏剧性 促进02-释放，而15-HETE抑制它。我们建议5- HETE通过增加细胞内钙离子的流量来启动中性粒细胞。 质膜，可能是通过为阳离子打开一个通道。这将是 测定这些细胞摄取45Ca~(2+)的速率 不同浓度的5-HETE。各种不同因素的影响 钙通道拮抗剂对这一过程将进行评估。这个 ~(45)Ca~(2+)吸收速率与02-产生的关系 将被定义，并且15-HETE可能的拮抗作用将是 被追寻。 关于蛋白质的磷酸化，我们观察到刺激 中性粒细胞释放02-总是伴随着强烈的 两种分子量约为47和49的蛋白质的磷酸化 KDA。从数量上讲，这是两种主要的蛋白质 在这些细胞的刺激下被磷酸化。而47 kDa的蛋白质 已经被广泛地描述了，几乎对49个 KDA种。实验在此详细描述以确定、表征 并提纯这种蛋白质。生物化学与细胞技术 生物学将会被使用。我们将确定49 kDa的蛋白质是否 02-生成系统的组件，或涉及其他一些 在刺激这些细胞的机制中流行(例如， 磷脂D)。治疗后49 kDa蛋白活性的变化 将寻求磷酸化来建立这种修饰之间的联系 反应和细胞刺激。

The long term objectives of this proposal are to elucidate the exact sequence of biochemical events that are involved in the priming and stimulation of superoxide (02-) production by neutrophils. Superoxide is a key component of the oxygen-dependent antimicrobial mechanisms of these cells which provide a major defense against infectious organisms. Certain cytokines prime neutrophils to release increased amounts of 02-. Therapeutic value of these factors is now being tested clinically in a variety of cancer and AIDS patients with promising results. Thus, experiments outlined herein are relevant to an understanding of host- defense mechanisms and immune deficiency diseases. Specifically, this project addresses two largely unknown areas. These are: (1) the mechanism(s) of action of the hydroxylated eicosatetraenoic acids (HETE) in modulating 02- production, and (2) the link between protein phosphorylation and 02- generation. Neutrophils produce 5- and 15-HETE under various circumstances. 5-HETE dramatically potentiates 02- release, whereas 15-HETE inhibits it. We propose that 5- HETE primes neutrophils by increasing the flux of Ca2+ across the plasmalemma, perhaps by opening a channel for this cation. This will be measuring the rates of 45Ca2+ uptake by these cells in the presence of different concentrations of 5-HETE. The effects of a variety of calcium channel antagonists on this process will be evaluated. The relationships between the rates of 45Ca2+ uptake and 02- generation will be defined, and possible antagonistic effects of 15-HETE will be sought. With regard to protein phosphorylation, we have observed that stimulation of 02- release by neutrophils is always accompanied by an intense phosphorylation of two proteins with molecular weights of ca. 47 and 49 kDa. Quantitatively, these are the two major proteins which are phosphorylated upon stimulation of these cells. While the 47 kDa protein has been extensively characterized, virtually nothing is known of the 49 kDa species. Experiments are detailed herein to identify, characterize and purify this protein. Techniques of biochemistry and cell biology will be employed. We will determine whether the 49 kDa protein is a component of the 02- generating system, or is involved in some other fashion in the mechanism of stimulation of these cells (e.g., phospholipose D). Changes in the activity of the 49 kDa protein after phosphorylation will be sought to forge a link between this modification reaction and cell stimulation.